A non-canonical multisubunit RNA polymerase encoded by the AR9 phage recognizes the template strand of its uracil-containing promoters

Abstract

AR9 is a giant Bacillus subtilis phage whose uracil-containing double-stranded DNA genome encodes distant homologs of β and β’ subunits of bacterial RNA polymerase (RNAP). The products of these genes are thought to assemble into two non-canonical multisubunit RNAPs - a virion RNAP (vRNAP) that is injected into the host along with phage DNA to transcribe early phage genes, and a non-virion RNAP (nvRNAP), which is synthesized during the infection and transcribes late phage genes. We purified the AR9 nvRNAP from infected B. subtilis cells and characterized its transcription activity in vitro. The AR9 nvRNAP requires uracils rather than thymines at specific conserved positions of late viral promoters. Uniquely, the nvRNAP recognizes the template strand of its promoters and is capable of specific initiation of transcription from both double- and single-stranded DNA. While the AR9 nvRNAP does not contain homologs of bacterial RNAP α subunits, it contains, in addition to the β and β’-like subunits, a phage protein gp226. The AR9 nvRNAP lacking gp226 is catalytically active but unable to bind to promoter DNA. Thus, gp226 is required for promoter recognition by the AR9 nvRNAP and may represent a new group of transcription initiation factors.

Multisubunit RNA Polymerases of Jumbo Bacteriophages

“…During the past two decades, extensive genome sequencing revealed genes coding for distant homologs of cellular RNAPs in the genomes of some eukaryotic viruses, bacteriophages, prophages and likely mobile elements located in the genomes of some Firmicutes and Cyanobacteria , and in fungal killer plasmids [ 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. Some of these genes were shown to encode functional RNAPs [ 23 , 24 , 25 , 26 , 27 , 28 ], while the products of others remain uncharacterized. These partially characterized and non-characterized putative RNAPs are collectively referred to as “non-canonical RNAPs” since they are highly divergent from multisubunit RNAPs of cellular organisms.…”

“…Though no vRNAP has been purified to date, nvRNAPs encoded by PBS2, phiKZ, and AR9 phages have been obtained and characterized to different extents [ 26 , 28 , 35 , 36 ]. The phiKZ nvRNAP was shown to consist of four predicted phage proteins jointly comprising the full-length β’- and β-like subunits and a fifth subunit gp68 with no sequence similarity to functionally characterized proteins [ 28 ].…”

“…Similarly to the phiKZ nvRNAP, the AR9 nvRNAP consists of β’ and β homologs and the fifth protein gp226 (a homolog of phiKZ gp68) [ 26 ]. In contrast to phiKZ nvRNAP it was possible to purify the four-subunit AR9 nvRNAP core complex composed of β’ and β homologs only [ 26 , 83 ]. The AR9 nvRNAP is catalytically active but cannot initiate transcription from promoters [ 26 ].…”

“…In contrast to phiKZ nvRNAP it was possible to purify the four-subunit AR9 nvRNAP core complex composed of β’ and β homologs only [ 26 , 83 ]. The AR9 nvRNAP is catalytically active but cannot initiate transcription from promoters [ 26 ]. The initiation-competent AR9 nvRNAP holoenzyme contains the fifth protein, gp226, which is thus formally equivalent to bacterial RNAP σ factors despite the lack of sequence similarity.…”

Global Proteomic Profiling of Salmonella Infection by a Giant Phage

“…As a result, a small C-terminal domain of the giant phage vRNAP ␤= was previously not detected and was only recently identified and confirmed to be essential (10,15). The main consequence of the two RNAPs is that giant phage gene expression programs are likely to have a high degree of independence from their host transcriptional machinery (18,(24)(25)(26).…”